Production of functional proteins: balance of shear stress and gravity

ABSTRACT

The present invention provides a method for production of functional proteins including hormones by renal cells in a three dimensional co-culture process responsive to shear stress using a rotating wall vessel. Natural mixture of renal cells expresses the enzyme 1-a-hydroxylase which can be used to generate the active form of vitamin D: 1,25-diOH vitamin D3. The fibroblast cultures and co-culture of renal cortical cells express the gene for erythropoietin and secrete erythropoietin into the culture supernatant. Other shear stress response genes are also modulated by shear stress, such as toxin receptors megalin and cubulin (gp280). Also provided is a method of treating in-need individual with the functional proteins produced in a three dimensional co-culture process responsive to shear stress using a rotating wall vessel.

RELATED APPLICATIONS

The present application claims the benefit of 35 U.S.C. §111(b)provisional patent application 60/043,205 filed Apr. 8, 1997.

ORIGIN OF THE INVENTION

Federal Funding Notice

The present invention was funded by NIH Grant DK46117, NIH R21, and NASANRA Grant 9-811. Consequently, the United States government has certainrights in this invention.

The jointly made invention described herein was made by an employee ofthe United States Government and may be manufactured and used by or forthe Government of the United States of America for governmental purposeswithout the payment of any royalties hereon or therefor.

The invention described herein was also made by inventors in theperformance of work under an agreement with Tulane Educational Fund andis subject to the provisions of Section 305 of the National Aeronauticsand Space Act of 1958, Public Law 85-568 (72 Stat. 435; 42 U.S.C. 2457).

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to the fields of proteinchemistry, endocrinology and gene therapy. More specifically, thepresent invention relates to a method for production of functionalproteins in culture in response to shear stress using a rotating wallvessel.

2. Description of the Related Art

A successful and documented modality to induce polarization anddifferentiation of cells in culture is the rotating wall vessel (1-4).In rotating wall vessels gravity is balanced by equal and oppositephysical forces including shear stresses. In engineering terms this hasbeen claimed to simulated microgravity at boundary conditions [Wolf D.A. and R. P. Schwarz. (1991) NASA Technical Paper 3143].

Rotating wall vessels, including models with perfusion, are a quantumadvance. The rotating wall vessel is a horizontally rotated cylindricalcell culture device With a coaxial tubular oxygenator (1, 5-7). Therotating wall vessel induces expression of select tissue-specificproteins in diverse cell cultures (1-2,8-9). Examples of expression oftissue-specific proteins include carcinoembryonic antigen expression inMIP-101 colon carcinoma cells (2), prostate specific antigen inductionin human prostate fibroblasts (7), through matrix material inductionduring chondrocyte culture (8). The quiescent cell culture environmentof the rotating wall vessel balances gravity with shear and other forceswithout obvious mass transfer tradeoff (1-2, 4). The rotating wallvessel provides a culture environment suitable for co-cultures ofdiverse cell types, and three dimensional tissue construct formation.

Rotating wall vessel technology is being used in clinical medicalpractice recently by facilitating pancreatic islet implantation (4, 10).Pancreatic islets are prepared in rotating wall vessels to maintainproduction and regulation of insulin secretion. The islets are alginateencapsulated to create a non-inflammatory immune haven, and areimplanted into the peritoneal cavity of Type I diabetic patients. Thisimplantation of pancreatic islets has maintained normoglycemia for 18months in diabetic patients, and progressed to Phase III clinical trials(4, 10). These vessels have also been applied to, for example, mammalianskeletal muscle tissue, cartilage, salivary glands, ovarian tumor cells,and colon crypt cells (11-13). Previous studies on shear stress responsein endothelial cells, and rotating wall vessel culture have been limitedto structural genes (14-16). These studies did not address the issue ofa process for the production of functional molecules, such as hormones.Shear stress response elements have not previously been demonstrated inepithelial cells, either for structural genes of production offunctional molecules.

Vitamin D dependent rickets has been a disease familiar to family farmsand larger animal husbandry industries for centuries (17-18). Thedevelopment of renal replacement therapy by dialysis in humans expandedvitamin D deficient bone disease from an occasional human clinicalcaveat to a common clinical problem. This led to identification of theactive form of vitamin D as 1,25-diOH D₃ and the development of amulti-billion dollar per year worldwide market, predominantly inend-stage renal disease patients, to provide replacement hormoneclinically (18). The active 1,25-diOH form of vitamin D₃ is mainly usedto treat bone disease in dialysis patients but has *also been implicatedas a therapy for osteoporosis, and some forms of cancer. Recently, theeffects of vitamin D have been recognized to play a central role notonly in other common bone lesions such as osteoporosis due to aging andsteroid induced osteoporosis, but in immune function and surveillance,growth and development, and cardiac and skeletal muscle function(19-22).

Several-active forms of vitamin D have been identified, vitamin Dreceptors cloned, and nuclear binding proteins for the hormoneidentified and cloned (17-22). Studies on the regulation of1α-hydroxylase activity are limited by the lack of a renal cell linewith regulated expression of the enzyme. The only reports of1-α-hydroxylase activity in culture utilize freshly isolated chickenrenal cortical cells in which the activity declines precipitously within48 hours of plating in culture (28).

The importance of the renal 1-α-hydroxylase is best understood bycomparing the kinetics of the renal enzyme to other forms in the body(29-30). Demonstration that nephrectomy in pregnant rats did notcompletely abolish 1,25-diOH-D₃ formation sparked an intensive searchfor extrarenal sites of 1 α-hydroxylase activity (29). Although 1α-hydroxylase activity has been reported in monocytes, liver, aorticendothelium and a variety of placental and fetal tissues, the enzymekinetics contrast sharply with the renal 1 α-hydroxylase. Extrarenal1-α-hydroxylase has a much higher Km indicating that much highersubstrate levels are needed for activity (29). In the uremic patient,extrarenal 1,25-diOH D₃ production is very limited due to a relativelack of substrate. Administrating large quantities of 25-OH D₃ substrateto anephric patients modestly boosts plasma 1,25-diOH D₃ levels (29).

The lack of a differentiated polarized line of renal tubular epithelialcells for investigative purposes persists despite extensive searches byseveral laboratories (31-38). Renally derived cell lines transformedwith viruses or tumor cells to produce immortality continue as some ofthe most popular cell biological tools to study polarized delivery (31,33, 35). But these renally derived immortal cell lines such as MDCK orLLP-CK1 retain few if any of the differentiated features characteristicof renal epithelial cells. Similarly, primary cultures rapidlydedifferentiate and modalities as diverse as basement membrane matrices,growth supplements or Millipore inserts achieve only modest degrees ofpolarity (37-38).

The pathognomonic structural features of renal proximal tubularepithelial cells are the abundance of apically derived microvilli, theglycoprotein content of associated intermicrovillar clefts, and thehighly distinctive arrangement of subapical endosomal elements (39-40).Renal epithelial cells of the proximal tubule are characterized bythousands of long apical microvilli. The apical endosomal machinerybegins in intermicrovillar clefts. The endosomal pathway ischaracterized by clathrin coated vesicles, small spherical endosomalvesicles, with deeper larger endosomal vacuoles (33, 39). From theendosomal vacuoles proteins and lipids either recycle to apical surfacein dense apical tubules or shuttle to lysosomes to be degraded.

A cluster of apical proteins with homologous sequence repeats areespecially desirable to express in cultured cells as they are thought tobe molecular mediators of renal injury (41-43). Two of these proteinsmegalin (gp330) and cubulin (gp280) (Moestrup, et al., J. Biol. Chem.β273 (9):5325-5242 (1998) are molecular mediators of tubular vacuolationand ensuing secondary damage. Megalin (gp330) is a receptor found on theluminal surface of the proximal tubular cells of the kidney. Megalinbinds several proteins and drugs including aminoglycoside antibioticsand other polybasic drugs. Megalin is expressed in the kidney, lung,testes, ear, and placenta. The only cells which express megalin inculture are immortalized placental cells. There is no known renal cellculture which expresses megalin. Gp280 is a receptor found on theluminal surface of the proximal tubular cells of the kidney. Gp280 bindsseveral proteins and drugs including intrinsic factor-cobalamin (vitaminB12 bound to its carrier protein) and myeloma light chains. Cubulin(gp280) is expressed in the kidney, ear, and placenta. The only cellswhich express cubulin (gp280) in culture are immortalized placentalcells. There is no known renal cell culture which expresses cubulin(gp280).

Erythropoietin (EPO) is a hormone produced in the kidney, and secretedinto the blood. Erythropoietin controls the rate of production of redblood cells by the bone marrow. Erythropoietin may be produced by theinterstitial cells between the tubules or the proximal tubular cells orboth. Erythropoietin production is lost in all known renal cell culturesystems. Erythropoietin is mainly used to treat anemia in dialysispatients but is also popular to treat the anemia of AIDS patients andmany forms of cancer.

The prior art is deficient in the lack of effective means of producingfunctional proteins including hormones in response to shear stress.Further, the prior art is deficient in the identification of shearstress response elements in epithelial cell genes. The present inventionfulfills this longstanding need and desire in the art.

SUMMARY OF THE INVENTION

In one embodiment of the present invention, there is provided a methodof producing a functional protein, comprising the steps of: isolatingmammalian cells; placing said cells into a rotating wall vesselcontaining a cell culture comprising culture media and culture matrix;producing three-dimensional cell aggregates under simulated microgravityconditions; and detecting expression of the functional protein in thecell culture.

In another embodiment of the present invention, there is provided amethod of inducing expression of at least one gene in a cell, comprisingthe steps of: contacting said cell with an transcription factor decoyoligonucleotide sequence directed against a nucleotide sequence encodinga shear stress response element; and determining the expression of saidgene in said cell.

In yet another embodiment of the present invention, there is provided atranscription factor decoy, comprising an oligonucleotide sequencedirected against a nucleotide sequence encoding a shear stress responseelement.

Other and further aspects, features, and advantages of the presentinvention will be apparent from the following description of thepresently preferred embodiments of the invention given for the purposeof disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

So that the matter in which the above-recited features, advantages andobjects of the invention, as well as others which will become clear, areattained and can be understood in detail, more particular descriptionsof the invention briefly summarized above may be had by reference tocertain embodiments thereof which are illustrated in the appendeddrawings. These drawings form a part of the specification. It is to benoted however, that the appended drawings illustrate preferredembodiments of the invention and therefore are not to be consideredlimiting in their scope.

FIG. 1 shows homogeneity and structure of human renal epithelial cellsin culture. Flow cytometry frequency histograms demonstrate number ofcells positive for the proximal tubular marker γ-glutanyl transferase.FIG. 1A shows the number of cells with y-glutamyl transferase activityas the frequency of activity in 2000 cells compared to an unstainedcontrol with trapping agent alone. This is the raw digest of human renalcells. FIG. 1B shows that following differential trypsinization, thepercentage of proximal tubular cells present can be increased to 99±1%.FIGS. 1C and 1D show transmission electron micrographs of humanepithelial cells in culture. The intact renal cortex in vivo (far leftpanel), is compared to culture of the natural mixture of human renalcortical cells in conventional 2-dimensional culture (middle left panel)which is completely devoid of microvilli. Rotating wall vessel cultureof pure proximal tubular cells shows some microvilli (middle rightpanel) but there are far more microvilli during rotating wall vesselculture of the natural mix of renal cortical cells (far right panel).Compared to these representative images, some areas of the naturalmixture of cells in the rotating wall vessel show much greater abundanceof microvilli, and well defined desmosomes (lower panel) which arelacking in the other cultures.

FIG. 2 shows protein expression in the rotating wall vessel. FIG. 2Ashows analysis of the expression and endosomal compartmentation ofmegalin, and cubulin in renal cells following rotating wall vesselculture. The ability of flow cytometry to make simultaneous measurementsof entrapped fluorescein dextran as an endosomal marker and antibodybinding allows construction of three dimensional frequency histogramsdisplaying entrapped fluorescein dextran fluorescence against antibodybinding on horizontal axes. A control sample shows vesicles negative forfluorescein on the left and fluorescein containing endosomes on theright (2000 vesicles depicted left panel). A control without fluoresceinentrapped shows only the left population (not shown). Co localization ofanti-cubulin binding demonstrates that all the fluorescein positiveendosomes are positive for cubulin, while non-endosomal membranes can besubdivided into cubulin positive and negative populations (middlepanel). This pattern is repeated for anti-megalin binding in renalcortical cells (right panel).

FIG. 2B shows quantitation of cubulin, and megalin antibody binding torenal cell membranes under various culture conditions. Analysis ofprotein expression in cultured cells by antibody binding used classicserial log dilution antibody curves. An increase in binding with adecrease in dilution is pathognomonic for specific antibody bindingduring flow cytometry analysis. Binding of anti-cubulin antisera tomembrane vesicles prepared from renal cortical cells after 16 days inculture, detected by the fluorescence of a phycoerthyrein taggedsecondary antibody, shows an almost two log increase in binding withantibody dilution (upper left panel below). This increased cubulinantibody binding in the cells grown in the rotating wall vessel (STLV)is more than five times the expression seen in stirred fermentors.Similarly, there was no detectable expression in the conventionalcultures resulting in a flat line (not shown). Binding of normal serumand minimal dilution of primary antisera were not detectably different.Binding curves for anti-megalin antiserum showed a similar pattern (notshown).

FIG. 2C depicts non-specific (minimum) and peak binding of eachantiserum following rotating wall vessel culture and two-dimensionalSDS-PAGE analysis of protein content of cells following rotating wallvessel culture. Analysis of the protein content of cultures of thenatural mixture of rat renal cortical cells after 16 days culture in gaspermeable bags as a control (left panel) or rotating wall vessel (rightpanel) depicts changes in a select set of proteins. Molecular weight(14-220 kDa) on the abscissa is displayed against isoelectric point (pH3-10) on the ordinate.

FIG. 3 shows gene expression in the rotating wall vessel. FIG. 3A andFIG. 3B show differential display of genetic expression of rat renalcortical cells grown in conventional culture or rotating wall vessels.Differential display of expressed genes was compared in aliquots of thesame cells grown in a 55 ml rotating wall vessel (STLV) or conventionalgas permeable 2-dimensional bag controls. For differential display,copies of expressed genes were generated by polymerase chain reactionusing random 25 mer primers and separated on a 6% DNA sequencing gel(FIG. 3A). Bands of different intensity between control and STLV,representing differentially expressed genes, were identified by visualinspection, excised and reamplified using the same primers. Differentialexpression and transcript size were confirmed by Northem hybridization(FIG. 3B). PCR products were then subcloned into the pGEM-T vector andsequenced. Sequences were compared to the Genebank sequences using theBLAST search engine. One expressed gene which decreased in the STLV(band D on gelabove) was identified as rat manganese-containingsuperoxide dysmutase (98% match 142 of 144 nucleotides). Two genes whichincreased in the STLV, band A was identified as the interleukin-1 betagene (100% match for 32 of 32 nucleotides) and Band B which correspondedto a 20 kB transcript on a Northern blot appears to be a unidentifiedgene that has a 76% homology to the mouse GABA transporter gene. FIG. 3Cand FIG. 3D show RT-PCR of time dependent change in genes duringrotating wall vessel culture. Semi quantitative RT-PCR shows increasesin the epithelial genes megalin, villin and extra-cellular calciumsensing receptor (ECaR), the shear stress response element genes ICAM,VCAM and MnSOD (FIG. 3C). There was no change in b-actin or GADPH.Unlike in endothelial cells many of these changes are prolonged as at 16days megalin, ECaR, ICAM, VCAM and villin changes persist (FIG. 3D).

FIG. 4 shows structure and effects of antisense probe for shear stressresponse element on rat renal cortical epithelial cells. FIG. 4A showsthe structure. The probe with sequence CTGAGACCGATATCGGTCTCAG (SEQ IDNo:1) has two possible conformations. As a single strand it would foldback on itself to form a binding element for the transcription factor.As a double strand it would then have two binding sites for thetranscription factor, one in the sense orientation and one in theantisense orientation.

FIG. 4B shows effects of antisense shear stress response element probeon time dependent gene expression. The antisense probe added toconventional 2-dimensional cultures of rat renal cortical cells at 80 nmincreases MnSOD in a time dependent manner. Comparison is made tocontrols with the active binding site scrambled. In contrast the probehas no effect on villin gene expression.

FIG. 5 shows gene expression in the rotating wall vessel: automated geneanalysis. Abundance of the expression of over 18,300 genes was assayedby annealing poly A RNA from human renal cortical epithelial cells grownin a rotating wall vessel for 8 days to a filter robotically loaded witholigonucleotide primers. Poly A RNA from a non adherent bag cultureserves as a control. The filters are shown at the top of the diagramthen the analysis of shear stress responsive genes, renal epitheliumspecific genes, and other genes germane to the current analysis.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to a method of producing a functionalprotein, comprising the steps of: isolating mammalian cells; placingsaid cells into a rotating wall vessel containing a cell culturecomprising culture media and culture matrix; producing three-dimensionalcell aggregates under simulated microgravity conditions; and detectingexpression of the functional protein in the cell culture. Generally,simulated microgravity conditions comprise a balance between gravity andoppositely directed physical forces. Representative examples suchphysical forces include sedimentational shear stress, centrifugalforces, viscosity and coriolus forces.

Preferably, the functional protein is selected from the group consistingof a hormone, a toxin receptor and a shear stress dependent functionalbiomolecule. Representative examples of hormones which can be producedaccording to the method of the present invention include1,25-dihydroxy-vitamin D3 and erythropoietin. Representative examples oftoxin receptors which can be produced according to the method of thepresent invention include megalin and cubulin. Representative examplesof shear stress dependent functional biomolecule which can be producedaccording to the method of the present invention include is selectedfrom the group consisting of villin, magnesium dependent superoxidedismutase, nitric oxide synthase, c-fos, c-jun, platelet derived growthfactor-b, transforming growth factor-b, tissue-type plasminogenactivator and monocyte chemotactic protein-1, megalin, cubulin,erythropoietin and 1-a-hydroxylase.

Generally, any mammalian cell could be used in the methods of thepresent invention. Representative examples of mammalian cells includerenal cortical cells, renal fibroblast cells, hepatocytes, pancreaticislets, renal interstitial cells, parathyroid cells, thyroid cells,pituitary cells, ovarian cells and testicular cells. Generally, the cellis selected from the group consisting of epithelial cell and endothelialcell. Preferably, cell contains shear stress response elements.Representative examples of shear stress response element include GAGACCand GGTCTC.

In the methods of the present invention, the rotating wall vessel isinitiated and maintained from about 6 rotations per minute to about 16rotations per minute. Preferably, the sedimentational shear stress isfrom about 0.2 dynes/cm2 to about 1.0 dynes/cm2. The culture matrix maycontain a core structure selected from the group consisting of cellaggregates and microcarrier beads, although other components to such aculture matrix are well known to those having ordinary skill in thisart.

The present invention is also directed to a method of inducingexpression of at least one gene in a cell, comprising the steps of:contacting said cell with an transcription factor decoy oligonucleotidesequence directed against a nucleotide sequence encoding a shear stressresponse element; and detennining the expression of said gene in saidcell. Generally, oligonucleotide comprises a terminal phosphothioratemoiety and a phosphodiester backbone and a structure which allows theoligonucleotide to pass cell membranes and accumulate in the nuclearcompartment of the cell. Generally, the cell is a cultured cell.Preferably, the cell is selected from the group consisting of anepithelial cell and an endothelial cell. Representative examples ofwhich can be used in this method include renal cortical cell, renalfibroblast cell, hepatocyte, pancreatic islet, renal interstitial cell,parathyroid cell, thyroid cell, pituitary cell, ovarian cell andtesticular cell. In one embodiment, the cell is grown in two dimensionalculture. Representative examples of shear stress response elementsinclude GAGACC and GGTCTC. Preferably, the gene encodes a proteinselected from the group consisting of megalin, cubulin, erythropoietinand 1-a-hydroxylase. The concentration of the oligonucleotide useful inthis method generally ranges from about 10 nm to about 10 mm.

The present invention is also directed to a transcription factor decoy,comprising an oligonucleotide sequence directed against a nucleotidesequence encoding a shear stress response element. Preferably, thenucleotide sequence encoding a shear stress response element has asequence selected from the group consisting of GAGACC and GGTCTC.

In one preferred technique, the rotating wall vessel is generallyinitiated and maintained at 10 rotations per minute. Preferably, therotating wall vessel provides a balance of forces comprising gravity andequal and opposite sedimentational shear stress. Useful sedimentationalshear stress rates within the context of the claimed methods are fromabout 0.2 dynes/cm2 to 1.0 dynes/cm2.

As used herein, rotating wall vessels refers to a cylidrical horizontalrotating culture vessel with a coaxial oxygenator.

As used herein, shear stress response element÷ refers to a sequence of afamily of genes in the cell nucleus which binds one or moretranscription factors in response to shear stress on the cell. Arepresentative example of a shear stress response element is GAGACC orits complementary sequence GGTCTC.

As used herein, shear stress conditions÷ refers to flow of liquid, orcurrent of liquid over cells which causes genes to turn on or off.

As used herein, slow turning lateral vessel÷ refers to one specific sizeand shape of a rotating wall vessel.

As used herein, differential display÷ refers to displaying on a filter,gel or chip a discrete set of genes turned on or off in a cell under twodifferent conditions.

As used herein, simulated microgravity÷ refers to balance of gravity byoppositely directed forces including shear stresses during rotationalwall vessel culture.

As used herein, graded gravitational sedimentation shear÷ refers to theshear imparted to a particle or cell falling through fluid.

As used herein, functional protein÷ refers to a protein with biologicaleffects.

As used herein, three-dimensional co-culture process÷ refers to cellsgrown in a matrix or on beads (or other three-dimensional structuralsuport) in a three-dimensional array, rather than on a flat plate.

As used herein, coriolus force÷ refers to an incidental flow fieldcaused by the rotating gravity vector in the rotating wall vessel.

As used herein, shear stress÷ refers to the force felt at the surface ofthe particle as it moves through the fluid.

As used herein, gravity induced sedimentation÷ refers to the force on aparticle in the rotating wall vessel making it fall through the fluiddue to gravity.

As used herein, centrifugal force÷ refers to the force on a particle inthe rotating wall vessel which pulls it towards the wall due torotational speed.

As used herein, transcription factor decoy÷ refers to an oligonucleotidefolded to form a double stranded DNA which binds a nuclear transcriptionfactor. The transcription factor decoy prevents the transcription factorfrom binding promoter regions regulating expression of specific genes.

The following examples are given for the purpose of illustrating variousembodiments of the invention and are not meant to limit the presentinvention in any fashion.

EXAMPLE 1

Human Renal Cortical Cells

Human renal cortical cells were isolated by Clonetics Inc. (San Diego,Calif.) from kidneys unsuitable for transplantation. Differentialtrypsinization resulted in cell fractions highly purified for proximaltubular cells compared to the natural mixture of cells in the renalcortex. The co-culture of the natural cell mix, and highly purifiedproximal tubular cells were cultured separately in a special growthmedium with 2% fetal calf serum.

EXAMPLE 2

Rat Renal Cortical Cells

Rat renal cells were isolated from renal cortex harvested fromeuthenized Sprague Dawley rats (Harlan Sprague-Dawley, Cleveland Ohio)as described (44). In brief, renal cortex was dissected out withscissors, minced finely in a renal cell buffer 137 mmol NaCl, 5.4 mmolKCl, 2.8 mmol CaCl₂, 1.2 mmol MgCl2, 10 mmol HEPES-Tris, pH 7.4. Theminced tissue was placed in 10 ml of a solution of 0.1% Type IVcollagenase and 0.1% trypsin in normal saline. The solution wasincubated in a 37° C. shaking water bath for 45 minutes withintermittent titration. The cells were spun gently (800 rpm for 5minutes), the supernatant aspirated, the cells resuspended in 5 ml renalcell buffer with 0.1% bovine serum, and passed through a fine (70 mm)mesh. The fraction passing through the mesh was layered over adiscontinuous gradient of 5% bovine serum albumin and spun gently. Thesupernatant was again discarded. The cells were resuspended in DMEM/F-12medium (ciprofloxacin and fungizone treated) and placed into culture invarious culture vessels in a 5% CO2 95% O2 incubator.

EXAMPLE 3

Culture Techniques: Rotating Wall Vessels

When grown under conventional conditions in DMEM/F12 supplemented withfetal calf serum and an antibiotic cocktail such as ciprofloxacin andfungizone, both the highly purified cells as well as the cell mix form amonolayer. Fetal calf serum was used at optimal concentration: 2% forhuman calls and 10% for rat cells. In order to increase epithelial celldifferentiation (1, 45), renal cells were cultured in a rotating wallvessels known as a 55 ml slow turning lateral vessel (STLV) (1, 45). Toinitiate cell culture, the slow turning lateral vessel was filled withmedium, and seeded by addition of cell suspension (2×106 cells/ml).Residual air was removed through a syringe port and vessel rotation wasinitiated at 10 rotations per minute, and maintained for 10-16 days.Medium was changed every 2 to 3 days depending on glucose utilization.Concomitant with cells, microcarrier beads were added an 5 mg/ml topromote aggregate formation in the slow turning lateral vessel. Withoutbeads the cells became shattered in the vessel in a few hours. Beadswere cytodex-3 in all protocol except when electron microscopy wasplanned when the much more expensive, but easily sectioned CultisphereGL cells were added to the vessels.

EXAMPLE 4

Stirred Controls and Static Controls

To provide a stirred control stirred fermentors which mixed in thehorizontal plane were loaded with identical concentrations of cells andbeads from the same pool added to the slow turning lateral vessel (1,31, 46). Gas permeable Fluoroseal bags (Fluoroseal Inc, Urbana Ill.) in7 or 55 ml size were selected as conventional static controls. Culturebeads were added to the conventional controls at the same density as theslow turning lateral vessel cultures (1, 45).

EXAMPLE 5

Electron Microscopy Quantitation of Number of Microvilli

Transmission electron micrographs were performed on cell aggregates fromthe rotating wall vessels and conventional monolayers. Cells were washedwith ice cold phosphate buffered saline, then fixed for electronmicroscopy with 2.5% glutaraldehyde in phosphate buffered saline (9,47). The samples were then transferred to 1% osmium tetroxide in 0.05 Msodium phosphate (pH 7.2) for several hours, dehydrated in an acetoneseries followed by embedding in Epon. Lead-stained thin sections wereexamined and photographed using a Phillips EM/200 electron microscope.For electron microscopy the easily sectioned Cultispere GL beads,replaced Cytodex-3 which is almost impossible to section.

EXAMPLE 6

Analysis of the Proximal Tubule Epithelial Marker, g-glutamylTranspeptidase

The renal cortical cells were 75+4% (n=4) proximal tubules as determinedby flow cytometry analysis of aliquots for the proximal markerg-glutamyl transferase using Schiff base trapping of cleavage productsof L-g-glu-4-methoxy-4-b-naphthylamine (44) (FIG. 1).

EXAMPLE 7

Analysis of the Endosomal Distribution of Megalin and Cubulin by FlowCytometry

To quantitate the total and endosomal expression of cubulin, megalin andaquaporin-2 cells in conventional culture, stirred fermentors and slowturning lateral vessel rotating wall vessels, 0.3 mg/ml 10Sfluorescein-dextran was to each cell culture for 10 minutes at 37° C. inthe CO2 incubator. This loads an entrapped fluorescent dye into theearly endosomal pathway (9, 47). Cells were then immediately dilutedinto ice cold phosphate buffered saline and washed once. Next, the cellswere homogenized with 6 passes of a tight fitting glass-Teflon motordriven homogenizer. A post-nuclear supernatant was formed as the 11,000g supernatant, 180,000 g pellet of membrane vessels (FIG. 2A).

Aliquots of membrane vesicles were labeled with megalin or cubulinantisera. The megalin and cubulin antisera were rabbit polyclonalsraised to affinity purified and chromatographically pure receptor (43,48). Membrane vesicles were first pre-incubated in 50% normal goat serumfor 2 hours to reduce non-specific binding of secondary antisera raisedin goat. After washing aliquots of membrane vesicles were stained withserial log dilution of antisera and incubated at 4° C. overnight. Afterfurther washing 1:40 of goat anti-rabbit affinity purified ratpre-absorbed phycoerthyrein conjugated secondary antiserum was added,and incubated for 4 hours at room temperature. Prior to flow cytometrythe membrane vesicles were washed and resuspended in 200 mM mannitol,100 mM KCl, 10 mM HEPES, pH 8.0 with Tris to which had been added 10 mMnigericin. In the presence of potassium, nigericin collapses pHgradients, ensuring optimal fluorescence of the highly pH dependentfluorescein-dextran emission. Fluorescein-dextran and antibody stainingtagged by phycoerythrein were now analyzed and co-localized on avesicle-by-vesicle basis by flow cytometry (FIG. 2B).

EXAMPLE 8

Differential Display

Differential display of expressed genes was compared in aliquots of thesame cells grown in a 55 ml rotating wall vessel (slow turning lateralvessel) or conventional gas permeable 2-dimensional bag controls (FIGS.3A and 3B). Differential display was performed using Delta RNAFingerprinting system (Clontech labs, Palo Alto Calif.). Copies ofexpressed genes were generated by polymerase chain reaction using random25 mer primers and separated on a 6% DNA sequencing gal. Bands ofdifferent intensity between control and slow turning lateral vessel,representing differentially expressed genes, were identified by visualinspection, excised and reamplified using the same primers. Differentialexpression and transcript size were confirmed by Northern hybridization.PCR products were then subcloned into the pGEM-T vector (Promega,Madison Wis.) and sequenced using fMOL cycle sequencing system (Promega,Madison, Wis.). Sequences were compared to the Genebank sequences usingthe BLAST search engine (National Center for Biotechnology Information).For genes of interest the bands were labeled with 32P for confirmationof the changes by Northern blot analysis.

EXAMPLE 9

Detection of Gene Expression in Cell Cultures by RT-PCR

Cell aggregates from the rotating wall vessel culture were washed oncein ice cold phosphate buffered saline and snap frozen at −70° C. untilRNA was isolated. Total RNA was first isolated, followed by isolation ofpoly A+ RNA. Following reverse transcription, 10%-20% of each cDNA wasamplified (Robocycler 40, Stratagene, La Jolla, Calif.) using 95° C.denaturation, 63° C. annealing and 72° C. extension temperatures.Amplification was for a total of 30 cycles with the first three cycleshaving extended denaturation and annealing times. Positive and negativestrand PCR primers, respectively, were derived from published sequencesusing Genetunner software. 20% of the PCR reaction was electrophoresedon agarose/ethidium bromide gels and visualized under UV light so that acomparison of amplified gene fragments could be made to DNA standards(HaeIII digested X174 DNA, Promega) electrophoresed on the same gel(FIGS. 3C and 3D). Representative fragments amplified for each gene inquestion were isolated from gels and direct sequenced to assure identityof the PCR product. In addition, 5% of the same cDNA were subjected toPCR for expression of the housekeeping mRNA, glyceraldehyde 3-phosphatedehydrogenase, and b-actin to assure that similar amounts of input RNAand that similar efficiencies of reverse transcription were beingcompared. Each cDNA was run in at least three dilutions to ensure thatmeasurements were made on the initial linear portion of the responsecurve.

EXAMPLE 10

Genetic Decoys

Double stranded genetic decoys matching the sequence of a known shearstress response element were synthesized (Chemicon International Inc.,La Jolla, Calif.) (structure and sequence shown at top of FIG. 4). Thesedecoys had a terminal phosphothiorate moiety to prevent intracellularlysis, and a phosphodiester backbone to facilitate passage across cellmembranes (49). Passage to and accumulation in the nuclear compartmentof cultured cells was confirmed by confocal imaging of a fluoresceintagged decoy. Three decoys were synthesized: the active decoy, a randomsequence control in which the six bases of the shear stress responseelement were scrambled, and a fluorescein conjugated form of the decoy.Decoys were placed in the cell culture medium of rat renal corticalcells grown as above in conventional two-dimensional culture. Aliquotsof cells exposed to control or active sequence decoy at 80 nmconcentration were harvested at 2, 6 and 24 hours after exposure.

EXAMPLE 11

Genetic Discovery Array

A sample of human renal cortical cells grown in conventional flaskculture was trypsinized and split into a gas permeable bag control and arotating wall vessel (55 ml slow turning lateral vessel). After 8 daysof culture on 5 mg/ml cytodex-3 beads, cells were washed once with icecold phosphate buffered saline, the cells were then lysed and mRNA wasselected with biotinylated oligo(dT) then separated with streptavidinparamagnetic particles (PolyATtract System 1000, Promega Madison, Wis.).32P labeled cDNA probes were then generated by reverse transcriptionwith 32P dCTP. The cDNA probes were hybridized to identical GeneDiscovery Array Filters (Genome Systems Inc. St. Louis, Mo.). The GeneDiscovery Array filters contain 18,394 unique human genes from theI.M.A.G.E. Consortium [LLNL](15) cDNA Libraries which are roboticallyarrayed on each of a pair of filter membranes. Gene expression was thendetected by phosphor imaging and analyzed using the Gene DiscoverySoftware [Genome Systems] (50).

EXAMPLE 12

Assay of 1-a-hydroxylase Activity

As the 1-a-hydroxylase enzyme has never been isolated or cloned it isassayed functionally by the production of 1,25-dihydroxy-vitamin D3 fromultrapure exogenous 25-hydroxy vitamin D3. For each measurement, theclassic. Michaelis Menton kinetics of the enzyme are determined byassaying equal aliquots of renal cell aggregates in a curve of 25-OH D3substrate concentrations from 0.1 to 10 mg/ml in 6 steps. Allincubations are performed in the presence of the anti-oxidant DPED at 10mM to ensure no contribution of non-enzymatic oxygenation (23-26).1,25-diOH D3 generated in vitrobwas quantitated as described (23-27). Invitroβincubations were terminated by adding a volume of acetonitrileequal to the incubation volume. Each incubation tube received 1,000 cpmof 3H-1,25 dihydroxy D3 to estimate recovery losses during the extensiveextraction and purification scheme. The 1,25-dihydroxy D3 is extractedfrom the incubation medium by C18 solid-phase extraction (24-25).Following extraction, the samples are evaporated to dryness under N2 anddissolved in 2 ml of methylene chloride. The samples are then applied tosilica Bond-Elut cartridges and the 1,25-dihydroxy D3-containingfraction is isolated and collected (26). The individual fractionscontaining 1,25-diOH D3 and then subjected to normal phase HPLC on aBeckman model 344 liquid chromatography system. Normal-phase HPLC wasperformed with a Zorbax-Sil column (26) (4×25 cm) developed in andeluted with methylene chloride/isopropanol (96:4 v/v) with a flow rateof 2 ml/min. The 1,25-dihydroxy D3 eluted from this system was driedunder N2 resuspended in ethanol and quantitated by radio receptor assayor radio immunoassay (25-26). Plasma 1-25-dihydroxy vitamin D3 wasassayed in a similar fashion, but as the product is already formed,assay begins with extraction into acetonitrile (23-26). Hence, allmeasurement of 1-a-hydroxylase activity in cells included determinationof the Michaelis Menton Km and Vmax, of the enzyme. The Michaelis Mentonparameters were determined by automated curve fitting.

EXAMPLE 13

Culturing Renal Fibroblasts and Assay for Production of Erythropoietin

As renal fibroblasts are the source of erythropoietin secreted into thecirculation, renal fibroblasts were cultured. Freshly dissected ratrenal cortex was minced and collagenase\trypsin digested prior toremoval of debris on a single discontinuous 5% albumin gradient. Themixture of rat renal cortical cells was placed into culture in DMEMF12with 20% fetal bovine serum. After two weeks to encourage fibroblastovergrowth in the rich medium, fibroblast growth factor was added. Theresultant culture had fibroblastic features in the culture flask and wasinoculated into a high aspect rotating vessell (HARV) for culture underincreased shear stress conditions. The cells aggregate on the beads andslowly increasing their numbers. After 3 weeks growing the fibroblastsin a HARV, erythropoietin was assayed in the cell supernatant. The mediawere concentrated 15× and assayed via RIA. The media alone was alsoconcentrated 15× as the control.

EXAMPLE 14

Culturing Hapatocytes and Assay for Production of Erythropoietin

As hepatocytes are a source of erythropoietin secreted into thecirculation, immortalized human hepatocyes were cultured under controland applied shear stress conditions. The Hep3B cells were placed intoculture in DMEM with 10% fetal bovine serum in static flask culture. Theresultant culture was split, one half remaining in static flask cultureand the other half was inoculated into a HARV for culture underincreased shear stress conditions. The cells aggregated on the beads.After 24 hours growing the Hep3B cells in a HARV, erythropoietin wasassayed in the cell supernatant. The media were assayed by RIA. Thestatic flask media was also assayed as the control.

EXAMPLE 15

Shear Stress Response Elements Mediate Changes in Erythropoietin GeneExpression

The immortal hepatic cell line, Hep3B, constitutively produceserythropoietin. The 5′ promoter and 3′ enhancer regions of the genecontain putative shear stress response elements. The role of theseelements in the enhancement of erythropoietin production in response toshear was tested by using integrated perfused rotating wall vesselculture to reintroduce graded shear. This protocol utilizes a library ofpromoters driving luciferase reporters genes, with various constructslacking the putative shear stress response elements. It also allows DNTAfootprinting analysis of the histones which bind the promoter andenhancer elements.

EXAMPLE 16

Results

The proportion of proximal tubular cells in human renal cell fractionsisolated by differential trypsinization was assayed using an entrappedfluogenic substrate for the proximal enzyme markerg-glutamyl-transferase (44). Flow cytometry analysis on a cell-by-cellbasis showed the natural cell mixture in the human renal cortex to be85+4%, n=4 proximal tubular cells (FIG. 1A, left panel). Followingdifferential trypsinization, and selection of the pure fractions,proximal tubular enrichments as high as 99+1% could be achieved (rightpanel). As reported in other systems, rotating wall vessels wereconducive to vigorous cell growth, as evidenced by the high rates ofglucose consumption assayed as 30 mg/dl glucose/100,000 cells/day. Acell doubling time of 4+3 days was assayed using Alamar blue in therotating wall vessel compared to 4+2 days in conventional culture (n=4).

The ultrastructure of cultures of pure proximal tubular cells or renalcortical cell mixtures of human kidneys were grown in rotating wallvessels for 16 days, and were examined by transmission electronmicroscopy (FIGS. 1B and 1C). Quantitation of the number of microvillipresent by counting random plates at the same magnification demonstratesnot only that the rotating wall vessel induces microvillus formation,but co-culture with the normal mix of renal cortical cells increases theeffect (Table 1). Normal cortical cell mix in conventionaltwo-dimensional culture has 2 1 microvilli per field; “pure” proximaltubular culture in rotating wall vessel has 10 4 microvilli per field;and the normal cortical cell mix. in rotating wall vessel has 35 11microvilli per field. TABLE 1 Human proximal tubular cells microvillicounted on transmission electron- micrographs of cells grown for 16 daysunder various culture conditions % Proximal Microvilli Per CultureConditions Tubular Markers Field conventional 2-D culture 85  2 1 pure÷culture in 99  10 4 rotating wall vessel normal cortical cell mix 85 3511 in rotating wall vessel

To examine the expression of megalin and cubulin in renal cells inculture, there are advantages to using human cells instead of rat cells.Specifically, the rat sequences of megalin and cubulin have been cloned,while the human sequences have not, and the antisera recognizes the ratbut not the human isoforms of these proteins. Hence, the natural mixtureof cells in the rat renal cortex was placed into culture in rotatingwall vessels, stirred fermentors, and traditional culture for analysisof protein expression.

As the endosomal pathway has been implicated to play a central role inthe function and pathophysiology of cubulin and megalin, entrappedendosomal markers were co-localized with receptor antibody binding. Theability of flow cytometry to make simultaneous measurements of entrappedfluorescein dextran as an endosomal marker and antibody binding allowsconstruction of three dimensional frequency histograms displayingentrapped fluorescein dextran fluorescence against antibody binding onhorizontal axes and number of vesicles in each channel up out of thepage (FIG. 2A). A control sample shows vesicles negative for fluoresceinon the left and fluorescein containing endosomes on the right (200vesicles depicted, left panel). A control without fluorescein entrappedshows only the left population (not shown). Co localization ofanti-cubulin binding demonstrates that all the fluorescein positiveendosomes were positive for cubulin, while non-endosomal membranes couldbe subdivided into cubulin positive and negative populations. (middlepanel). This pattern was repeated for anti-megalin binding in renalcortical cells (right panel) in culture.

Next, analysis of protein expression in cultured cells by antibodybinding used classic serial log dilution antibody curves. An increase inbinding with a decrease in dilution is pathognomonic for specificantibody binding during flow cytometry analysis. Binding of anti-cubulinantisera to membrane vesicles prepared from renal cortical cells after16 days in culture, detected by the fluorescence of a phycoerthyreintagged secondary antibody, shows an almost two log increase in bindingwith antibody dilution (FIG. 2B). This increase in the cells grown inthe rotating wall vessel (slow turning lateral vessel) is more than fivetimes the expression seen in stirred fermentors. Similarly there was nodetectable expression in the conventional cultures resulting in a flatline (not shown). Comparison of maximal binding of the anti-cubulinantibody to minimum taken to be the antibody dilution at which there isno further decline in signal with primary antibody dilution is shown inFIG. 2C. Binding of normal serum and minimal dilution of primaryantisera were not detectably different. Binding curves for anti-megalinantiserum showed a similar pattern (not shown) but the peak binding wasa little lower (FIG. 2C). Again stirred fermentor has much lessexpression than the rotating wall vessel (slow turning lateral vessel)and the conventional cell membranes have no detectable binding (notshown).

To examine the proportion of proteins changing in the rotating wallvessel, two-dimensional gel SDS-PAGE analysis on cultures grown in therotating wall vessel and bag controls were performed (FIG. 2 d). Theresults shown in FIG. 2D demonstrates changes were in a selected groupof proteins.

To identify the genes changing during rotating wall vessel culture,differential display were performed. Differential display of expressedgenes was compared in aliquots of the same cells grown in a 55 mlrotating wall vessel (slow turning lateral vessel) or conventional gaspermeable 2-dimensional bag controls. Differential display of copies ofexpressed genes were generated by polymerase chain reaction using random25 mer primers and separated on a 6% DNA sequencing gel. Bands ofdifferent intensity between control and slow turning lateral vessel,representing differentially expressed genes, were identified by visualinspection, excised and reamplified using the same primers. Differentialexpression and transcript size were confirmed by Northern hybridization.PCR products were then subcloned into the pGEM-T vector and sequenced.Sequences were compared to the Genebank sequences using the BLAST searchengine. One expressed gene which decreased in the slow turning lateralvessel (band D on gel, FIG. 3A) was identified as ratmanganese-containing superoxide dismutase (98% match 142 of 144nucleotides). Two genes which increased in the slow turning lateralvessel, band A was identified as the interleukin-1 beta gene (100% matchfor 32 of 32 nucleotides) and Band B which corresponded to a 20 kBtranscript on a Northern blot appears to be a unidentified gene that hasa 76% homology to the mouse GABA transporter gene.

To examine the genetic changes in specific genes, the expression oftissue specific epithelial cell markers and classic shear stressresponse dependent genes were examined by RT-PCR (FIG. 3 c). Severalgenes specific for renal proximal tubular epithelial cells, includingmegalin, cubulin, the extracellular calcium sensing receptor, and themicrovillar structural protein villin, increase early in rotating wallvessel culture. Similarly there were dynamic time dependent changes inclassic shear stress dependent genes including intercellular adhesionmolecule 1 (ICAM) and vascular cell adhesion molecule (VCAM) (increased)and manganese dependent superoxide dismutase (suppressed). Many but notall of these changes were prolonged, as after 16 days in culture geneexpression of megalin, ICAM, VCAM and the extracellular calcium sensingreceptor were still elevated, while villin and manganese dependentsuperoxide dismutase were at control levels. Expression of controlGADPH, b-actin and 18S genes did not change throughout the time course.

To test for a role of a putative endothelial shear stress responseelement in these renal cortical cell changes, an antisense probe for thesequence was synthesized (FIG. 4A). A control probe had the active motifscrambled. Confocal imaging of a fluorescein conjugated form of theprobe confirmed nuclear delivery of the probe (images not shown).Culture of rat renal cortical cells in 80 nm of the probe, resulted in atime dependent increase in magnesium dependent superoxide dismutase, butno change in villin gene expression (FIGS. 4B and 4C). The control probehad no effect.

In order to confirm the genetic responses to rotating wall vesselculture and the analysis with human cells, automated gene displayanalysis of expressed RNA was performed on human renal cortical cellsgrown in a control gas-permeable bag and the slow turning lateral vesselfor 8 days (50). Of the more than 18,000 genes assayed a select groupwas again observed to change (FIG. 5). In particular, vectored changesin all the classic shear stress response genes assayed by RT-PCR anddifferential display in rat cell culture were confirmed. A battery oftissue specific genes was increased including villin, angiotensinconverting enzyme, parathyroid hormone receptor and sodium channels.Other physical force dependent genes such as heat shock proteins 27/28kDa and 70-2 changed, as did focal adhesion kinase, and a putativetranscription factor for shear stress responses NF-kb changed. Fusionproteins such as synabtobrevin 2 mildly decreased gene expression, andclathrin light chains hugely increased gene expression.

To determine whether renal cells grown in simulated microgravity have1-a-hydroxylase activity, the 1-a-hydroxylase activity of cell cultureswere compared grown in traditional 2-D culture in gas permeable bags,and NASA rotating wall vessels. Both rat renal cells (Table 2) and humanembryonic renal cells were assayed (Table 3). TABLE 2 The 1a-hydroxylase activity of the various rat renal cell cultures detectedas production of 1,25-diOH D3 1,25-diOH D3 concentration Volume of1,25-diOH D3 Cell Sample (pg/ml) Supernatant (ml) Production (pg) Boiledstatic <2, not detectable 7 ml Not detectable control I Boiled static<2, not detectable 7 ml Not detectable control II Static control I <2,not detectable 7 ml Not detectable Static control II <2, not detectable7 ml Not detectable Boiled rotating <2, not detectable 55 ml  Notdetectable wall vessel Rotating wall 14.2 55 ml  781 vessel

The results shown in TABLE 2 indicate that rat renal cells showincreased structural differentiation during culture in simulatedmicrogravity conditions, and express much greater 1-a-hydroxylaseactivity than under conventional culture conditions. TABLE 3 The 1a-hydroxylase activity of the various human embryonic renal cellcultures detected as production of 1,25-diOH D3 1,25-diOH D3Concentration Volume of 1,25-diOH D3 Cell Sample (pg/ml) Supernatant(ml) Production (pg) Boiled static control 8.2 10 82 Static control 14.610 146 Rotating wall vessel 24.8 55 1364

TABLE 3 indicates that human embryonic kidney cells show increasedstructural differentiation during culture in simulated microgravityconditions, and express 10 foid greater 1-a-hydroxylase activity thanunder conventional culture conditions. TABLE 4 Renal fibroblasts cellsupernatant erythropoietin assay Condition Erythropoietin (mu/ml) Shearstress culture 1.8 Control media conc 15X 0.23

TABLE 5 Hepatocytes cell supernatant erythropoietin assay ConditionErythropoietin (mu/ml) Shear stress culture 141.7 mu/1 × 106 cellsControl static flask undetectable

Results of cell supernatant erythropoietin assay from renal fibroblastsand hepatocytes culture were shown in Table 4 and Table 5, respectively.The results shown in TABLES 4 and 5 indicate erythropoietin productionwas increased in both renal and hepatic cells during gradedgravitational sedimentation shear.

Erythropoietin has the classic shear stress response elements in thepromoter and enhancer regions which control expression of its gene. Theresults shown in Tables 4 and 5 also indicate that the expression of theerythropoietin gene was upregulated by those shear stress responseelements during graded gravitational sedimentation shear in the vessel.

EXAMPLE 17

Discussion

Rotating wall vessels have been used by other investigators as“simulated microgravity”. The present invention contends that gravity isstill active, and that in a rotating wall vessel gravity is balanced byequal and opposite sedimentational shear stress. A centrifugal force dueto spinning the cells, quantitatively much smaller than gravity, is alsopresent and offset by equal and opposite sedimentational shear stress.Thus, the present invention presents a new concept that rotating wallvessels provide this new balance of forces, including application ofsedimentational shear, rather than microgravity.

The rotating wall vessel bioreactor provides quiescent co-localizationof dissimilar cell types (1, 46), mass transfer rates that accommodatemolecular scaffolding and a micro-environment that includes growthfactors (1, 46). Engineering analysis of the forces active in the vesselis complex (1, 5-7). This study provides the first evidence for the cellbiological mechanisms by which the vessel induces changes in tissuespecific gene and protein expression.

There are two possible explanations why the rotating wall vessel inducesan order of magnitude more expression of the renal toxin receptorscubulin and megalin than stirred fermentor culture. First, there aresignificant differences in the degree of shear stress induced. Therotating wall vessel induces 0.5-1.0 dynes/cm2 shear stress (1), whilestirred fennentors induce 2-40 dynes/cm2 depending on design androtation speed (1, 5, 46). This degree of stress damages or kills mostepithelial cells (1, 5, 46). Second, impeller trauma in the stirredfermentor, is absent in the rotating wall vessel. This explains whythere was far more cubulin and megalin induced in renal cultures inrotating wall vessel culture than a stirred fermentor, and bothreceptors were not detectable in conventional 2-dimensional culture.

Rotating wall vessel culture induced changes in a select set of genes,as evidenced by the genetic differential display gels and 2-dimensionalprotein gel analysis. For example, erythropoietin production iscontrolled by a shear stress element which mediates changes observedduring graded gravitation sedimentation shear. 1-a-hydroxylase activityis maintained and increased in both renal cortical epithelial cells andhuman embryonic kidney cells, wherein the induction of the enzyme(1-a-hydroxylase) converts 25-hydroxy-vitamin D3 to the active1,25-dihydroxy-vitamin D3 form. The present invention is the firstdemonstration of a process for production of molecules includinghormones and other biomolecules induced by shear stress and otherforces. The mechanistic information can be interpreted from knowledge ofthe pattern of response and distribution of certain gene products.

Megalin and cubulin represent the first pattern of change, as theseproteins are restricted in distribution to renal cortical tubularepithelial cells. The increase in megalin mRNA and protein, and cubulinprotein expression is therefore unequivocal evidence for changes in theepithelial cells. This provides an important new tool for studies ofnephrotoxicity. Long suspected to play a role in renal toxicity, thetissue restricted giant glycoprotein receptors megalin and cubulin, haverecently been shown to be receptors for common nephrotoxins. Megalin isa receptor for polybasic drugs such as the aminoglycoside antibioticgentamicin (48) and vitamin D binding protein (51), and cubulin is thereceptor for vitamin-B12 intrinsic factor (52). Although these receptorsare expressed by transformed placental cells in culture (9, 43), thereis currently no renal model expressing these markers for toxicologyinvestigations (53). Rotating wall culture provides a fresh approach toexpression of renal specific markers in culture for study on thepharmacology, biochemistry and toxicology which define the uniqueproperties and sensitivities of renal epithelial cells.

The second pattern of change is represented by villin. Message for themicrovilli protein villin increases in the rotating wall vessel in thefirst day of culture, and soon reformation of microvilli was observed. Adecoy matching the nuclear binding motif of a putative shear stressresponse element failed to induce similar changes. Although the promoterfor villin has not been cloned, this suggests the changes in villin wereinduced by other transcription factors which may be due to shear stressor other stimuli in the bioreactor. Villin is also restricted to brushborder membranes such as renal proximal tubular cells, or colonic villi(54-55). The observed increases in villin message resolved after 16 daysof rotating wall vessel culture.

Magnesium dependent superoxide dismutase represents a third pattern ofresponse: a mitochondrial enzyme, ubiquitous is distribution, modulatedby the classic shear stress response element in endothelial cells(56-57). Magnesium dependent superoxide dismutase message decreasedearly in the first day of rotating wall vessel culture, and this waspersistent after 16 days in culture. These changes were confirmed whenmagnesium dependent superoxide dismutase was identified as suppressed inthe differential display analysis of gene changes, and Northern blotconfirmation was performed. A decoy for the classic shear stressresponse element induced an increase in magnesium dependent superoxidedismutase (MnSOD), which indicates that similar changes to the rotatingwall vessel can be induced by the use of genetic decoys. Thus, thebiological process of genetic induction by defined shear stress elementscan be produced by multiple means including genetic decoys or use of therotating wall vessel. Other shear stress response element dependentgenes, specifically, intercellular adhesion molecule 1 (ICAM) andvascular cell adhesion molecule (VCAM) had changes in the rotating wallvessel opposite to magnesium dependent superoxide dismutase, mirroringobservations made during flow induced stress in endothelial cells(56-57). This provides three lines of evidence consistent with a rolefor shear stress as one mediator of genetic changes induced in therotating wall vessel.

Differential display of the genes activated and deactivated underrotating wall vessel culture conditions showed rotating wall vesselculture was associated with decreased expression of manganese dependentsuperoxide dismutase mRNA and increased expression of interleukin-1 bgene mRNA. This greatly extends and brings together previousobservations on the interactions of stress, manganese dependentsuperoxide dismutase expression and interleukin-1. Topper et al.reported an oppositely directed effect, i.e., differential display ofvascular endothelial cells exposed to high stress demonstrates increasedmanganese dependent superoxide dismutase gene expression (57). Otherdirect evidence links superoxide dismutase and interleukin-1 asincreases in manganese superoxide dismutase levels and decreases ininterleukin-1 levels in HT-1080 fibrosarcoma cells (58). In moreindirect evidence overexpression of mitochondrial manganese superoxidedismutase promotes the survival of tumor cells exposed to interleukin-1(59). The present study provides direct evidence that modest shearstress decreases magnesium dependent superoxide dismutase in associationwith an inverse effect on interleukin-1.

The data here demonstrates internal consistency. The changes inmagnesium dependent superoxide dismutase were observed on differentialdisplay, confirmed by Northern blot analysis, and matched responses weredetected by RT-PCR. Megalin demonstrated matched changes in RT-PCR geneand protein expression. Changes in villin observed by RT-PCR wereassociated with dramatic reformation of microvilli, in which villin is amajor structural protein. Although semi-quantitative RT-PCR is prone toinherent variation due to the massive amplification of signals, the useof multiple controls which remain unchanged (b-actin, GAPDH and 18S),and experimental confirmation that reactions were linearly related tocDNA concentration, minimizes these problems. The internally consistentfindings by other methods strongly suggests this RT-PCR data is valid.

Study of the mechanisms of action of the rotating wall vessel to inducegene and protein expression during cell culture has been hampered bynomenclature. First, the attachment of the moniker “simulatedmicrogravity”, based on engineering analysis of boundary conditions,clouds intuitive analysis of the cell biology as there is no cellularequivalent for this term (1, 6-7). Similarly the reduced shear stress inthe rotating wall vessel compared to stirred fermentors leads to theterm “reduced shear stress culture” (1), whereas there is increasedshear stress compared to conventional 2-dimensional culture (1, 5). Ascell aggregates remain suspended in the rotating wall culture vessels,gravity is balanced by an equal and opposite force. Engineeringarguments on the relative contributions of fluid shear, drag,centrifugal force, coriolus motion, and tangential gravity-inducedsedimentation are themselves tangential to the cell biology. Severallines of evidence are documented that shear stress responses are one ofthe component of the biological response. This opens the door foranalysis of other biological response mediators in the vessels, andinvestigation as to whether unloading of gravity plays as big a role asthe oppositely directed balancing forces.

Using the rotating wall vessel as a tool, data here provide the firstevidence that shear stress response elements, which modulate geneexpression in endothelial cells, are also active in epithelial cells,although other investigators failed to see an effect of shear stress onepithelial cells. The present invention demonstrates that epithelialcells have shear stress response elements and change gene expression inresponse to physical forces including but not limited to sedimentationalshear stress. As the rotating wall vessel gains popularity as a clinicaltool to produce hormonal implants it is desirable to understandmechanisms by which it induces genetic changes (10, 60), if necessary toprolong the useful life of implants. Several lines of evidence areprovided that shear stress response elements are the first mechanismidentified by which the rotating wall vessel induces genetic changes.Using a putative endothelial cell shear stress response element bindingsite as a decoy, the role of this sequence in the regulation of selectedgenes in epithelial cells was validated. However, many of the changesobserved in the rotating wall vessel are independent of this responseelement. It remains to define other genetic response elements modulatedduring rotating wall vessel culture, and whether the induced changes aresecondary to the balancing forces, or primarily related to unloadinggravity.

The following references were cited herein.

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Any patents or publications mentioned in this specification areindicative of the levels of those skilled in the art to which theinvention pertains. These patents and publications are hereinincorporated by reference to the same extent as if each individualpublication was specifically and individually indicated to beincorporated by reference.

One skilled in the art will readily appreciate that the presentinvention is well adapted to carry out the objects and obtain the endsand advantages mentioned, as well as those inherent therein. The presentexamples along with the methods, procedures, treatments, molecules, andspecific compounds described herein are presently representative ofpreferred embodiments, are exemplary, and are not intended aslimitations on the scope of the invention. Changes therein and otheruses will occur to those skilled in the art which are encompassed withinthe spirit of the invention as defined by the scope of the claims.

1-12. (canceled) 13-26. (canceled) 27-35. (canceled)
 36. A method ofmonitoring renal tubular epithelial differentiation comprising: a)isolating at least one cell b) placing said cell into a rotating wallvessel containing a cell culture comprising culture media and culturematrix; and c) monitoring expression of greater than one gene in anarray, wherein the expression of said genes is indicative ofdifferentiated renal tubular epithelial cells.
 37. The method of claim27, wherein said gene is selected from the group consisting of1-α-hydroxylase, megalin, cubulin, erythropoietin, manganese super oxidedysmutase, interleukin-1β, a GABA transporter gene, β actin, villin,extracellular calcium sensing receptor, ICAM, VCAM, and γ-glutamyltransferase.
 38. The method of claim 27, wherein expression of a gene isincreased.
 39. The method of claim 29, wherein said gene is selectedfrom the group consisting of 1-α-hydroxylase, megalin, cubulin,erythropoietin, manganese super oxide dysmutase, interleukin-1β, a GABAtransporter gene, β actin, villin, extracellular calcium sensingreceptor, ICAM, VCAM, and γ-glutamyl transferase.
 40. The method ofclaim 27, wherein expression of a gene is decreased.
 41. The method ofclaim 31, wherein said gene is selected from the group consisting of1-α-hydroxylase, megalin, cubulin, erythropoietin, manganese super oxidedysmutase, interleukin-1β, a GABA transporter gene, β actin, villin,extracellular calcium sensing receptor, ICAM, VCAM, and γ-glutamyltransferase.
 42. A method of producing active renal epithelial cellscomprising: a) isolating renal stem cells; and b) culturing said cellsin a rotating wall vessel containing a cell culture comprising culturemedia and culture matrix.
 43. The method of claim 34 whereinshear-stress response is reduced by the addition of a transcriptionfactor decoy oligonucleotide encoding a shear-stress response elementspecific sequence.
 44. A method of producing active 1,25-dihydroxyvitamin D3 comprising: a) isolating at least one cell; b) placing saidcell into a rotating wall vessel containing a cell culture comprisingculture media and culture matrix; and c) inducing 1,25-dihydroxy vitaminD3 production.